CYP450 and drug efflux transporters polymorphism influence clinical outcomes of Thai osimertinib-treated non-small cell lung cancer patients

Background: Osimertinib has shown greater efficacy than standard epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) and fewer grade 3 or higher adverse drug reactions (ADRs) in patients with advanced non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations. However, the clinical outcomes of osimertinib treatment vary depending on the patient’s ethnicity. Therefore, further research is necessary to evaluate the impact of single nucleotide polymorphisms (SNPs) in cytochrome P450 (CYP450) and drug transporters on the therapeutic outcomes and ADRs to osimertinib in Thai patients, to provide improved pharmacological treatments for cancer patients. Methods: This retrospective and prospective cohort study enrolled 63 Thai patients with NSCLC treated with 80 mg of osimertinib once daily as monotherapy. Seventeen SNPs in candidate genes related to drug metabolism and transport pathways were analyzed in each patient. Chi-square or Fisher’s exact tests were used to evaluate the associations between SNPs and clinical outcomes, including ADR incidence and objective response rate (ORR). In addition, the correlation between the genotype and median time to treatment failure (TTF) or progression-free survival (PFS) was assessed using Kaplan-Meier analysis and a log-rank test. Results: We identified six SNPs (rs2231142 and rs2622604 in ABCG2, rs762551 in CYP1A2, rs1057910 in CYP2C9, rs28371759 in CYP3A4, and CYP2A6 deletion polymorphism (CYP2A6*4)) that significantly increased the incidence of ADRs. In addition, we found two SNPs (rs2069514 in CYP1A2 and rs1057910 in CYP2C9) that significantly decreased the median TTF, and two SNPs (rs28399433 in CYP2A6 and rs1057910 in CYP2C9) that significantly decreased the median progression-free survival (PFS). Specifically, we found that one of these SNPs (rs1057910 in CYP2C9) influenced ADRs, TTF, and PFS. Additionally, SNPs in the CYP2A6 heterozygous variant (non4/*4) significantly increased ADR incidence, leading to a high frequency of dose reduction (27.0%). Conclusion: Our study demonstrated significant SNPs associated with increased ADR incidence, decreased PFS, and decreased TTF in Thai patients with NSCLC treated with osimertinib. The CYP2C9 (*3) and CYP2A6 (*4) allele frequencies differed between ethnicities and were associated with an increased incidence of ADRs. These findings highlight the importance of considering genetic factors in NSCLC treatment and may facilitate personalized medicine approaches. Moreover, our study showed a higher incidence of ADRs than the previous trials, including FLAURA and AURA2, and a higher frequency of dose reduction than reported in the AURA 3 trial, possibly due to genetic differences among the study populations.


Introduction
Lung cancer is the leading cause of cancer-related deaths, accounting for approximately 18% (Sung et al., 2021).Approximately 80%-85% of these cases are classified as nonsmall cell lung cancer (NSCLC) (Nicholson et al., 2022).In Thai patients with NSCLC, epidermal growth factor receptor (EGFR) mutations were most commonly detected, accounting for 68% of cases (Detarkom et al., 2018).Activating EGFR mutations have been identified as predictive indicators of sensitivity to firstand second-generation EGFR tyrosine kinase inhibitors (TKIs).Acquired resistance develops 9-12 months after treatment initiation (Westover et al., 2018).One common mechanism underlying acquired resistance is the substitution of threonine with methionine at amino acid position 790 (T790M) in exon 20 of the EGFR gene, which accounts for 50%-60% of cases.This mutation impairs the binding of both firstand second-generation EGFR-TKIs by enhancing the ATP-binding affinity of the kinase domain of the EGFR mutant receptor, leading to treatment resistance (Yu et al., 2013).
In a previous study, variability in the steady-state area under the plasma drug concentration-time curve (AUCs) of AZ5104 between ethnic groups was observed, with a 10%-23% decrease in Asian versus Caucasian patients; however, the underlying reason remains unclear (Brown et al., 2017).CYP450 predominantly metabolizes Osimertinib and is a substrate for P-glycoprotein (P-gp), which is encoded by ABCB1, and breast cancer resistance protein (BCRP), which is encoded by ABCG2 (AstraZeneca, 2021), which may lead to individual variations in plasma osimertinib concentrations due to genetic polymorphisms.A previous study also found a linear relationship between ADR development and osimertinib

Clinical endpoint assessment
We analyzed the association between the SNPs and clinical outcomes, including the incidence of adverse drug reactions (ADRs), median time to treatment failure (TTF), median progression-free survival (PFS), and objective response rate (ORR).ADRs were periodically assessed by each patient's physician using the National Cancer Institute Common Terminology Criteria for Adverse Events version 5.0.The Naranjo algorithm was used to determine causality.TTF was the interval between initiating osimertinib treatment and a new locally directed or systemic treatment other than osimertinib monotherapy.PFS was defined as the time from osimertinib treatment initiation to disease progression or death from any cause.ORR was defined as the percentage of study patients who achieved a complete or partial response to treatment within a certain period, as assessed by each patient's physician according to the Response Evaluation Criteria in Solid Tumors version 1.1.

Statistical analysis
Associations between SNPs and clinical outcomes, including ADRs and ORR, and those between patients' baseline characteristics and clinical outcomes were evaluated using the appropriate chi-square test or Fisher's exact test.Genetic polymorphisms were assessed for concordance with Hardy-Weinberg Equilibrium (HWE) using Fisher's exact test.Linkage disequilibrium was explored using Haploview version 4.0.Univariate and multivariate logistic regression analyses were performed to identify the factors associated with clinical outcomes.The multivariate logistic regression analyses included all variables and all SNPs with a p-value of <0.1 from the univariate analysis, which is presented in Supplementary Table S2.The correlation between TTF, PFS, and genotype was assessed using Kaplan-Meier analysis and the log-rank test.Statistical analysis was performed using SPSS version 23 for Windows, and significance was set at p < 0.05.The sample size calculation was based on a prospective longitudinal observational cohort study of 53 patients with advanced NSCLC receiving osimertinib therapy, which found a significant association between SNPs rs2231137 in ABCG2 and grade ≥2 adverse events (p = 0.008).Of the ABCG2 wild-type (G/G) patients, 22 (68.75%) had grade ≥2 adverse events, while all three (100%) of the ABCG2 mutant-type (A/A) patients had grade ≥2 adverse events (Ishikawa et al., 2023).Therefore, based on the output of sample size calculation from n4Studies for a cohort study with binary outcomes (Ngamjarus, 2016), at least 58 patients were needed.

Genotype frequencies
The genotype status of drug-metabolizing enzymes and transporters was determined for all 63 patients (Table 2).No genotype distribution deviated from the Hardy-Weinberg equation.

SNPs associated with osimertinib efficacy outcomes
After initiating osimertinib, 31 patients (49.2%) had an objective response, including one patient (1.6%) with a complete response and The median TTF was 19 (10.3-29.0)months.In addition, SNPs rs2069514 in CYP1A2 mutant-type (A/A) and rs1057910 in CYP2C9 heterozygous variant (A/C) were significantly associated with decreased TTF with p < 0.001 and 0.041, respectively.These findings are presented in Table 5 and Figure 1.
The median PFS was significantly decreased in patients with SNPs rs28399433 in CYP2A6 mutant-type (C/C) and rs1057910 in CYP2C9 heterozygous variant (A/C), with p = 0.023 and <0.001, respectively.In addition, among patients who received osimertinib as second-line therapy (N = 36), SNPs rs28399433 in CYP2A6 mutant-type (C/C) and rs1057910 in CYP2C9 heterozygous variant (A/C) were also significantly associated with decreased PFS, with p values of 0.001 and 0.010, respectively (Table 6; Figure 2).

Discussion
A recent population pharmacokinetic study showed a linear correlation between exposure to osimertinib, measured using the AUCs of the parent compound and two active metabolites (AZ5104 and AZ7550), and the incidence of ADRs (Brown et al., 2017).AZ5104, in particular, demonstrated an 8-fold greater potency against EGFR mutations (Han et al., 2021).While AZ7550 and AZ5104 are present in approximately 10% of the parent compound (Brown et al., 2017), a decrease in AZ5104 AUCs of 10%-23% in Asian patients compared with that of Caucasian patients may influence clinical outcomes.In addition, a study in Asian populations reported a significant association between SNPs rs1128503 in ABCB1 and rs2231137 in ABCG2 and osimertinib-induced grade ≥2 adverse events (Ishikawa et al., 2023).However, the association between SNPs and the efficacy of osimertinib remains unclear, and the specific SNPs that influence the pharmacokinetics and clinical effects of osimertinib in NSCLC remain unknown.This is the first study to analyze a large number of genetic polymorphisms in candidate genes involved in the osimertinib pharmacokinetic pathway to enable the assessment of both efficacy and safety endpoints.All allele frequencies were consistent with the Hardy-Weinberg equilibrium (PharmGKB, 2021).In the present study, the frequency of dose reduction (27.0%) was higher than that reported in the AURA 3 trial (16.5%), and the incidence of ADRs was higher than that in the FLAURA, AURA2, and AURA3 trials (Goss et al., 2016;Mok et al., 2017;Soria et al., 2018).This may be because of genetic differences between patient populations; Asian patients who were administered osimertinib in these studies accounted for only 62%, 63%, and 65% of the patients, respectively.The frequency of the ABCG2 rs2231164 (C) allele, known as a loss-of-function variant, was 23.72% in the South Asian population and only 12.12% in the European population (Whirl-Carrillo et al., 2021).Furthermore, the frequency of the CYP2A6*4 allele, a slower metabolizer, also differed significantly between ethnic groups (Pang et al., 2015), potentially leading to higher plasma concentrations of osimertinib in the Thai population.These genetic differences may have contributed to the observed differences in dose reductions and ADRs between our study and previous clinical trials.Additionally, we found that the ORR in our study was 49.2%, which was lower than that reported in the AURA 3 trial (71%) (Mok et al., 2017).This difference in response rates may be because of the differences in patient populations between the two studies.In the AURA 3 trial,     .12 -ClinVar -NCBI, 2013).This variant may lead to poor metabolism of osimertinib in patients carrying the CYP2C9*3 allele, which increases the risk of osimertinibinduced ADRs due to higher osimertinib exposure but also decreases survival outcomes due to lower exposure to its active metabolite (AZ5104).These findings are similar to those of a previous study on the osimertinib exposure-response relationship, which found that the mortality rate was significantly higher in the high osimertinib drug level group (Rodier et al., 2022).Additionally, the probability of developing rashes, diarrhea, or QTc prolongation increased with exposure, and a linear relationship between adverse event development and osimertinib levels was identified (Brown et al., 2017).The mechanism that supports EGFR-TKI-induced ADRs is the inhibition of EGFR1 and EGFR2 (HER2) signaling, leading to a reduction in growth and impaired healing of the epithelium where EGFR is expressed.This subsequently causes alterations in keratinocyte proliferation and differentiation, reduced growth, impaired healing of the intestinal epithelium, and alterations in myocyte growth (Giovannini et al., 2009;Hirsh et al., 2014;Ikebe et al., 2020).
Our study has several limitations.First, the sample size was small, and some clinical data were retrospectively collected from medical records.Nevertheless, no genotype distribution deviated from the Hardy-Weinberg equation (PharmGKB, 2021), and the clinical data were confirmed by the patient's physician.Second, our inability to control for other confounding factors, such as compliance with osimertinib treatment caused by home oral medication but we had oncology pharmacist pill count measures of compliance at every visit, and all patients maintained a 100% compliance rate.Additionally, there were no observed drug interactions that influenced osimertinib drug levels.However, we did not restrict patient coffee consumption, which may have an impact on higher enzyme activity in the SNP rs762551 in CYP1A2 mutant-type (A/A).This enzyme was inducible by heavy coffee consumption and has been associated with a lower incidence of ADRs (Djordjevic et al., 2010;Wang et al., 2012).Finally, a pharmacokinetic analysis was not included in this study.However, the association between SNPs and clinical outcomes was consistent with that in previous reports.
In conclusion, our study identified significant SNPs associated with increased ADRs incidence, decreased TTF, and decreased PFS in Thai patients with NSCLC treated with osimertinib.The findings can potentially guide treatment decisions and help optimize individualized therapy for patients with NSCLC harboring EGFR mutations.However, more extensive studies with analysis of osimertinib and its active metabolite drug levels must be conducted to confirm these findings.The success of this study required the assistance and contributions of many individuals.We would like to express our sincere gratitude to all the patients who agreed to participate in this study and the staff who provided invaluable support throughout the research process.
rs1057910 (C), located in the CYP2C9 gene, typically encodes the amino acid leucine at position 359, and the resulting allele is also known as CYP2C9*3, which is a decreased function variant (VCV000008408

FIGURE 2
FIGURE 2 Kaplan-Meier estimates and log-rank tests for median progression-free survival (PFS) associated with SNPs among patients who received osimertinib as second-line therapy.(A) association between SNPs rs28399433 in CYP2A6 and PFS; and (B) association between SNPs rs1057910 in CYP2C9 and PFS.

TABLE 1
Baseline characteristics of the patients enrolled in the study.

TABLE 2
Genotype and allele frequencies of SNPs were compared between the present study and a previous report(PharmGKB, 2021).

TABLE 3
Genotype and allele frequencies of SNPs were compared between the present study and a previous report (PharmGKB, 2021) (cont.).

TABLE 4
Summary of significant association between SNPs and incidence of specific ADRs.
n, number of ADRs, in the genotype; N, number of all cases in the genotype; (%), incidence rate.a Statistically significant.

TABLE 6
Association between SNPs and clinical outcomes (cont.).
number of ADRs, in the genotype; N, number of all cases in the genotype; All grade ADRs, and Severity of ADRs (N = 63); TTF, median time to treatment failure (N = 20); PFS, median progression-free survival with osimertinib as second-line therapy (N = 36); 95% CI, 95% confidence interval; NE, not estimable.
a Statistically significant.

TABLE 7
Incidence of adverse drug reactions.